Release reagent for vitamin d compounds

ABSTRACT

A reagent composition for releasing vitamin D compounds bound to vitamin D-binding protein and an in vitro method for the detection of a vitamin D compound in which the vitamin D compound is released from vitamin D-binding protein by the use of this reagent composition as well as the reagent mixture obtained in this manner. Also disclosed is the use of the reagent compositions to release vitamin D compounds as well as a kit for detecting a vitamin D compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/874,702 filed Oct. 5, 2015, which is a continuation of U.S. patentapplication Ser. No. 14/277,359 filed May 14, 2014, which is acontinuation of International Application No. PCT/EP2012/072569 filedNov. 14, 2012, which claims the benefit of European Patent ApplicationNo. 11189736.9 filed Nov. 18, 2011, the disclosures of which are herebyincorporated by reference in their entirety.

BACKGROUND INFORMATION

The present invention concerns a reagent composition for releasingvitamin D compounds bound to vitamin D-binding protein, an in vitromethod for the detection of a vitamin D compound in which the vitamin Dcompound is released from vitamin D-binding protein by the use of thisreagent composition and the reagent mixture obtained in this manner. Italso concerns the use of the disclosed reagent composition to releasevitamin D compounds as well as a kit for detecting a vitamin D compoundwhich contains the reagent composition for releasing vitamin D compoundsin addition to common detecting reagents.

An adequate supply of vitamin D is vital as the term “vitamin” alreadysuggests. A deficiency of vitamin D leads to severe diseases such asrickets or osteoporosis. While vitamin D was still regarded as a singlesubstance at the beginning of the last century, the vitamin D system haschanged in the course of the last decades into a complex and manifoldnetwork of vitamin D metabolites. Nowadays more than 40 differentvitamin D metabolic products are known (Zerwekh, J. E., Ann. Clin.Biochem. 41 (2004) 272-281).

Humans can only produce D₃ vitamins or calciferols by the action ofultraviolet rays from sunlight on the skin. In the blood Vitamin D₃ isbound to the so-called vitamin D-binding protein and transported to theliver where it is converted into 25-hydroxyvitamin D₃ by25-hydroxylation. A multitude of other tissues are nowadays known to beinvolved in vitamin D metabolism in addition to the skin and liver, thetwo organs that have already been mentioned (Schmidt-Gayk, H. et al.(eds.), “Calcium regulating hormones, vitamin D metabolites and cyclicAMP”, Springer Verlag, Heidelberg (1990) pp. 24-47). 25-Hydroxyvitamin Dand more specifically 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ arethe central storage form of vitamin D in the human organism with regardto their amounts. When needed these precursors can be converted in thekidneys to form the biologically active 1α,25-dihydroxyvitamin D theso-called D hormone. The biologically active vitamin D regulates amongothers calcium uptake from the intestine, bone mineralization and itinfluences a large number of other metabolic pathways such as e.g. theinsulin system.

Measuring the vitamin D level itself is of little benefit whendetermining the vitamin D status of a patient, because concentrations ofvitamin D (vitamin D₂ and vitamin D₃) fluctuate greatly depending onfood uptake or exposure to sunlight. In addition vitamin D has arelatively short biological half-life in the circulation (24 hours) andit is therefore also for this reason not a suitable parameter fordetermining the vitamin D status of a patient. The same also applies tophysiologically active forms of vitamin D (1,25-dihydroxyvitamin D).These biologically active forms also occur in relatively small andhighly fluctuating concentrations compared to 25-hydroxyvitamin D. Forall these reasons the quantification of 25-hydroxyvitamin D inparticular is a suitable means to globally analyse the total vitamin Dstatus of a patient.

Vitamin D metabolites like 25-hydroxyvitamin D are bound with highaffinity by vitamin D-binding protein and to a limited extend also toalbumin and some lipoproteins. Methods appropriate to release a vitaminD metabolite from vitamin D-binding protein will under normalcircumstances also be more than appropriate to release a vitamin Dmetabolite also from any other protein.

The binding of 25-hydroxyvitamin D or other vitamin D compounds to thevitamin D-binding protein enormously complicates the determination ofvitamin D compounds. All known methods require that the vitamin Dcompound to be analysed is released or detached from the complex that itforms with the vitamin D-binding protein. In the following this isreferred to as the release of a vitamin D compound from vitaminD-binding protein for the sake of simplification although of course itcan only be released from a complex of vitamin D compound and vitaminD-binding protein and not from the vitamin D-binding protein alone.

The vitamin D-binding protein is unfolded at acidic pH but has a hightendency to correctly refold and to re-bind the analyte when the pH isshifted back to neutral conditions. Hence, it is often necessary tofirstly release vitamin D compounds and then to separate the vitaminD-binding protein from the vitamin D compounds to be analysed.

Due to the high clinical importance of 25-hydroxyvitamin D a largenumber of methods are known from the literature which allow25-hydroxyvitamin D to be more or less reliably determined.

Haddad, J. G. et al., J. Clin. Endocrinol. Metab. 33 (1971) 992-995, andEisman, J. A. et al., Anal. Biochem. 80 (1977) 298-305 for exampledescribe the determination of 25-hydroxyvitamin D concentrations inblood samples using high performance liquid chromatography (HPLC).

Other approaches for the determination of 25-hydroxyvitamin D are basedamong others on the use of vitamin D-binding proteins like those thatare present in milk. Thus Holick, M. F. and Ray, R. (U.S. Pat. No.5,981,779) and DeLuca et al. (EP 0 583 945) describe vitamin D assaysfor hydroxyvitamin D and dihydroxyvitamin D which are based on thebinding of these substances to vitamin D-binding protein where theconcentrations of these substances are determined by means of acompetitive test procedure. However, a prerequisite of this method isthat vitamin D metabolites to be determined firstly have to be isolatedfrom the original blood or serum samples and have to be purified by, forexample, chromatography.

Armbruster, F. P. et al. (WO 99/67211) teach that a serum or plasmasample should be prepared for vitamin D determination by ethanolprecipitation. In this method the protein precipitate is removed bycentrifugation and the ethanolic supernatant contains soluble vitamin Dmetabolites. These can be measured in a competitive binding assay.

Alternatively EP 0 753 743 teaches that the proteins can be separatedfrom blood or serum samples using a periodate salt. In this case vitaminD compounds are determined in the protein-free supernatant from thesamples treated with periodate. In some commercial tests acetonitrile isrecommended for the extraction of serum or plasma sample (e.g. in theradioimmunoassay from DiaSorin or in the vitamin D test from the“Immundiagnostik” Company).

In recent years a number of different release reagents were proposedwhich should in principle be suitable for releasing vitamin D compoundsfrom any binding protein present in the sample. However, this release ordetachment should be carried out under relatively mild conditions thusenabling a direct use of the sample treated with the release reagent ina binding test (see for example WO 02/57797 and US 2004/0132104).Despite immense efforts in recent years, all available methods fordetermining vitamin D have disadvantages such as laborious samplepreparation, poor standardization, poor agreement between testprocedures or bad recovery of spiked vitamin D (see for this inparticular Zerwekh, J. E., supra).

In U.S. Pat. No. 7,087,395 metal hydroxids as well as cyclodextrin andderivatives thereof, and metal salicylates have been used to releasevitamin D compounds from vitamin D-binding protein, which result in anirreversible denaturation of vitamin D-binding protein or other serumproteins. Surfactants like Triton X100 or Tween-20 have been used toprevent the vitamin D compound from being non-specifically attached tolipids and proteins in the sample after denaturation.

It is particularly difficult to automate a test for a vitamin Dcompound. The automation requires solving a very difficult problem i.e.surviving a tightrope walk: On the one hand it is necessary to releasethe vitamin D compounds from vitamin D-binding protein with the aid of asuitable release reagent, on the other hand, the conditions have to beselected such that the sample can be directly analysed further. Aprerequisite of this direct further analysis is that, on the one hand,the endogenous vitamin D-binding protein does not bind or no longer to asignificant extent binds to the vitamin D compounds during this analysisand thus does not interfere with this analysis and, on the other hand,that the release reagent used does not interfere with the binding ofdetection reagents such as antibodies, or vitamin D-binding protein. Inaddition it is known that different alleles of the vitamin D-bindingprotein are present in the human population which behave biochemicallydifferently. The release and measurement of vitamin D compounds shouldbe comparable for various alleles/phenotypes.

Thus the object of the present invention was to develop a reagentcomposition for release of vitamin D compounds and in particular forhydroxyvitamin D compounds from vitamin D-binding protein in a samplewhich can at least partially overcome the problems of the prior art. Asuitable reagent composition for releasing vitamin D compounds, an invitro method for determining vitamin D compounds the use of the reagentcomposition and kits for the determination of vitamin D compounds usingthis reagent composition are described in the following and areencompassed by the attached claims.

SUMMARY OF THE INVENTION

The present invention concerns an in vitro method for releasing avitamin D compound from vitamin D-binding protein comprising the step ofa) providing a sample to be investigated and b) mixing the sample fromstep (a) with i) a reagent containing one hydrogen carbonate salt and asubstance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) uponhydrolysis, wherein the total concentration of hydrogen carbonate ions(HCO₃ ⁻) from the hydrogen carbonate salt and released from thesubstance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) is 0.1 Mto 2.0 M, ii) a reducing agent, and iii) an alkalinising agent, therebyreleasing the vitamin D compound from vitamin D-binding protein.

In a further embodiment the present invention concerns an in vitromethod for measuring a vitamin D compound comprising the steps of a)providing a sample to be investigated, b) mixing the sample from step(a) with i) a reagent containing one hydrogen carbonate salt and asubstance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) uponhydrolysis, wherein the total concentration of hydrogen carbonate ions(HCO₃ ⁻) from the hydrogen carbonate salt and released from thesubstance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) is 0.1 Mto 2.0 M, ii) a reducing agent, and iii) an alkalinising agent, therebyreleasing a vitamin D compound from vitamin D-binding protein, and c)measuring the vitamin D compound released in step (b).

In a further embodiment the present invention concerns a reagentcomposition for the release of a vitamin D compound from vitaminD-binding protein comprising one hydrogen carbonate salt and a substancecapable of releasing hydrogen carbonate ions (HCO₃ ⁻) upon hydrolysis,wherein the total concentration of hydrogen carbonate ions (HCO₃ ⁻) fromthe hydrogen carbonate salt and released from the substance capable ofreleasing hydrogen carbonate ions (HCO₃ ⁻) is 0.1 M to 2.0 M and areducing agent.

In a further embodiment the present invention concerns a reagent mixturecomprising a sample to be investigated, a reagent composition for therelease of a vitamin D compound from vitamin D-binding proteincomprising one hydrogen carbonate salt and a substance capable ofreleasing hydrogen carbonate ions (HCO₃ ⁻) upon hydrolysis, wherein thetotal concentration of hydrogen carbonate ions (HCO₃ ⁻) from thehydrogen carbonate salt and released from the substance capable ofreleasing hydrogen carbonate ions (HCO₃ ⁻) is 0.1 M to 2.0 M and areducing agent, and an alkalinising agent.

In a further embodiment the present invention concerns a kit for therelease of a vitamin D compound from vitamin D-binding protein, whichcontains a reagent composition comprising one hydrogen carbonate saltand a substance capable of releasing hydrogen carbonate ions (HCO₃ ⁻)upon hydrolysis, wherein the total concentration of hydrogen carbonateions (HCO₃ ⁻) from the hydrogen carbonate salt and released from thesubstance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) is 0.1 Mto 2.0 M and a reducing agent.

DETAILED DESCRIPTION

The present invention concerns an in vitro method for releasing avitamin D compound from vitamin D-binding protein comprising the step ofa) providing a sample to be investigated, b) mixing the sample from step(a) with i) a reagent containing one hydrogen carbonate salt and asubstance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) uponhydrolysis, wherein the total concentration of hydrogen carbonate ions(HCO₃ ⁻) from the hydrogen carbonate salt and released from thesubstance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) is 0.1 Mto 2.0 M, ii) a reducing agent, and iii) an alkalinising agent, therebyreleasing the vitamin D compound from vitamin D-binding protein.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle.

The expression “one or more” denotes 1 to 50, preferably 1 to 20 alsopreferred 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 15.

If not stated otherwise the term “vitamin D compound” is to beunderstood to include all naturally occurring compounds which containthe backbone of vitamin D2 or the backbone of vitamin D3 according tothe following structural formulae I and II.

In the structural formulae I and II the positions of vitamin D arestated according to the steroid nomenclature. The 25-hydroxyvitamin Ddenotes vitamin D metabolites that are hydroxylated at position 25 ofthe structural formulae I and II i.e. the 25-hydroxyvitamin D₂ as wellas the 25-hydroxyvitamin D₃. Additional known hydroxyvitamin D compoundsare e.g. the 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D forms.

1,25-Dihydroxyvitamin D refers to the active forms of vitamin D (theso-called D hormones) that have a hydroxylation at position 1 as well asat position 25 of the structural formulae I and II.

Other well known vitamin D compounds are 24,25-dihydroxyvitamin D₂,24,25-dihydroxyvitamin D₃ and C3-epi 25-hydroxyvitamin D.

Surprisingly it has been found by the inventors, that the presence ofone hydrogen carbonate salt and a substance capable of releasinghydrogen carbonate ions (HCO₃ ⁻) upon hydrolysis under alkalineconditions in the in vitro method disclosed in the present inventionleads to the release of vitamin D compounds from vitamin D-bindingprotein.

A “hydrogen carbonate ion” (bicarbonate ion) according to the presentinvention is an anion with the empirical formula HCO₃ ⁻ and a molecularmass of 61.01 daltons.

A “hydrogen carbonate salt” according to the present invention is acompound selected from the group consisting of sodium hydrogen carbonate(NaHCO₃), potassium hydrogen carbonate (KHCO₃), ammonium hydrogencarbonate (NH₄HCO₃), calcium hydrogen carbonate (Ca(HCO₃)₂) andmagnesium hydrogen carbonate (Mg(HCO₃)₂.

A “substance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) uponhydrolysis” according to an embodiment of the present invention is acarbonate ester.

A “carbonate ester” according to the present invention is a carbonylgroup flanked by two alkoxy groups. The general structure of thesecarbonates is R₁O(C═O)OR₂. There are cyclic carbonate esters (e.g.ethylene carbonate) or non-cyclic carbonate esters (e.g. dimethylcarbonate) as well as hydroxylated or halogenized derivatives thereofavailable.

Preferably one hydrogen carbonate salt and a substance capable ofreleasing hydrogen carbonate ions (HCO₃ ⁻) upon hydrolysis according tostep i) of the method has a total concentration of 0.1 M to 1.5 M, or of0.2 M to 1.0 M, or of at least 0.1, 0.2, 0.3 or 0.4 M, or of at most2.0, 1.7, 1.5, 1.3, 1.0 or 0.75 M, respectively.

In a preferred embodiment the hydrogen carbonate salt is selected fromthe group consisting of sodium hydrogen carbonate, potassium hydrogencarbonate, ammonium hydrogen carbonate, calcium hydrogen carbonate andmagnesium hydrogen carbonate. In a further preferred embodiment thehydrogen carbonate salt is selected from the group consisting of sodiumhydrogen carbonate, potassium hydrogen carbonate and ammonium hydrogencarbonate. Further preferred the hydrogen carbonate salt is selectedfrom the group consisting of sodium hydrogen carbonate and potassiumhydrogen carbonate.

In a preferred embodiment the substance capable of releasing hydrogencarbonate ions (HCO₃ ⁻) upon hydrolysis is a cyclic or non-cycliccarbonate ester or a hydroxylated or halogenized derivative thereof,respectively. In a further preferred embodiment the substance capable ofreleasing hydrogen carbonate ions (HCO₃ ⁻) upon hydrolysis is a cyclicor non-cyclic carbonate ester or a halogenized derivative thereof,respectively. In a further preferred embodiment the cyclic or non-cycliccarbonate ester or the halogenized derivative thereof is selected fromthe group consisting of ethylene carbonate, dimethyl carbonate,propylene carbonate, vinylene carbonate, trimethylene carbonate,erythritol bis-carbonate, glycerol 1,2-carbonate,4-chloro-1,3-dioxolan-2-one, 4,5-dichloro-1,3-dioxolan-2-one,2,5-dioxahexanedioic acid dimethyl ester, 1,2 butylene carbonate, cis2,3 butylene carbonate and trans 2,3 butylene carbonate. Furtherpreferred the cyclic or non-cyclic carbonate ester or the halogenizedderivative thereof is selected from the group consisting of ethylenecarbonate, dimethyl carbonate, propylene carbonate, vinylene carbonate,trimethylene carbonate, erythritol bis-carbonate, glycerol1,2-carbonate, 4-chloro-1,3-dioxolan-2-one and4,5-dichloro-1,3-dioxolan-2-one.

Further preferred the cyclic or non-cyclic carbonate ester is selectedfrom the group consisting of ethylene carbonate, dimethyl carbonate,glycerol 1,2-carbonate, propylene carbonate and vinylene carbonate.Further preferred the cyclic or non-cyclic carbonate ester is selectedfrom the group consisting of ethylene carbonate, dimethyl carbonate,glycerol 1,2-carbonate and propylene carbonate. Further preferred thecyclic or non-cyclic carbonate ester is selected from the groupconsisting of ethylene carbonate, dimethyl carbonate and glycerol1,2-carbonate.

It is known to a person skilled in the art that one or more hydrogencarbonate salt(s) and one or more substance(s) capable of releasinghydrogen carbonate ions (HCO₃ ⁻) upon hydrolysis, respectively, can bearbitrarily mixed in order to achieve the effect disclosed in thepresent invention.

In an embodiment the reagent containing one hydrogen carbonate salt anda substance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) uponhydrolysis of step (i) according to the method of the present inventionis soluble to at least 2 M in an aqueous solution under the appropriateconditions for releasing a vitamin D compound from vitamin D-bindingprotein. In a further embodiment the reagent containing one hydrogencarbonate salt and a substance capable of releasing hydrogen carbonateions (HCO₃ ⁻) upon hydrolysis of step (i) according to the method of thepresent invention is soluble to at least 1.5 M, or more preferred issoluble to at least 1.0 M, in an aqueous solution under the appropriateconditions for releasing a vitamin D compound from vitamin D-bindingprotein. It is known to the skilled artisan how to solubilize onehydrogen carbonate salt and a substance capable of releasing hydrogencarbonate ions (HCO₃ ⁻) upon hydrolysis in water to achieve the reagentof step (i) of the method according to the present invention. Thehydrogen carbonate salt and the substance capable of releasing hydrogencarbonate ions (HCO₃ ⁻) upon hydrolysis should be soluble in water at25° C. The hydrogen carbonate salt and the substance capable ofreleasing hydrogen carbonate ions (HCO₃ ⁻) upon hydrolysis solubilizedin an aqueous solution should be storable at a temperature of 4° C.without drop out or crystallization.

The molar ratio of the hydrogen carbonate salt and the substance capableof releasing hydrogen carbonate ions (HCO₃ ⁻) upon hydrolysis to thealkalinising agent is preferably between 1:3 and 3:1, more preferablybetween 1:2 and 2:1 and more preferably between 1:1.5 and 1.5:1.

The skilled artisan is also aware, that the molar ratio of alkalinisingagent to hydrogen carbonate salt and/or substances capable of releasinghydrogen carbonate ions (HCO₃ ⁻) upon hydrolysis is calculated on thecorresponding concentrations of the reactive ions OH⁻ or HCO₃ ⁻.

It is known to the skilled artisan that a mixture of one hydrogencarbonate salt and a substance capable of releasing hydrogen carbonateions (HCO₃ ⁻) upon hydrolysis can be used in a method according to thepresent invention. The molar ratio of said mixtures of hydrogencarbonate salt and/or the substance capable of releasing hydrogencarbonate ions (HCO₃ ⁻) upon hydrolysis to the alkalinising agent ispreferably between 1:3 and 3:1, more preferably between 1:2 and 2:1 andmore preferably between 1:1.5 and 1.5:1.

Without wanting to be bound to this theory, it may well be that thepresence of one hydrogen carbonate salt and a substance capable ofreleasing hydrogen carbonate ions (HCO₃ ⁻) upon hydrolysis in thereagent composition induces a pH shift. Lower concentrations of saidhydrogen carbonate salt and/or said substance capable of releasinghydrogen carbonate ions (HCO₃ ⁻) upon hydrolysis in a reagentcomposition cause a slower pH reduction of the reagent mixture duringthe pre-treatment reaction. Higher concentrations of said hydrogencarbonate salt and/or said substance capable of releasing hydrogencarbonate ions (HCO₃ ⁻) upon hydrolysisin the reagent composition causea faster pH reduction of the reagent mixture during the pre-treatmentreaction. It also would appear that due to the concerted action of onehydrogen carbonate salt and a substance capable of releasing hydrogencarbonate ions (HCO₃ ⁻) upon hydrolysis and reducing agent at alkalinebuffer conditions an irreversible denaturation of vitamin D-bindingprotein is achieved and thereby later detection of a vitamin D compoundis facilitated.

In an embodiment the reducing agent of step ii) according to the methodis selected from the group consisting of 2-Mercaptoethanol,2-Mercaptoethylamine-HCl, TCEP, Cystein-HCl, Dithiothreitol (DTT),N-Methylmaleimide, Ellman's Reagent and 1,2-dithiolane-3-carboxylicacid.

In a further embodiment the reducing agent of step ii) according to themethod is characterized in that the reducing agent of step ii) containsthiol groups.

In a further embodiment the reducing agent of step ii) according to themethod is selected from the group consisting of 2-Mercaptoethanol,2-Mercaptoethylamine-HCl, TCEP, Cystein-HCl and Dithiothreitol (DTT).

In an embodiment the reducing agent of step ii) according to the methodhas a concentration from 2 mM to 30 mM, in a further embodiment from 3mM to 20 mM, in a further embodiment from 3.5 mM to 15 mM, and in afurther embodiment from 4 mM to 10 mM.

An “alkalinising agent” can be an alkali hydroxide or alkaline earthmetal hydroxide (i.e. in an aqueous solution). An alkalinising agent mayalso comprise a mixture of alkali hydroxides and/or alkaline earth metalhydroxides, i.e. NaOH and KOH, NaOH and LiOH, NaOH and Ca(OH₂), KOH andCa(OH)₂, KOH and LiOH, as well as other combinations.

“Alkali hydroxides” are a class of chemical compounds which are composedof an alkali metal cation and the hydroxide anion (OH—). Alkalihydroxides are such as NaOH, KOH, LiOH, RbOH and CsOH. “Alkali metals”are a series of chemical elements forming Group 1 (IUPAC style) of theperiodic table: lithium (Li), sodium (Na), potassium (K), rubidium (Rb),caesium (Cs), and francium (Fr).

“Alkaline earth metal hydroxides” are a class of chemical compoundswhich are composed of an alkaline earth metal cation and 2 hydroxideanions (OH—). “Alkaline earth metals” comprising Group 2 (IUPAC style)(Group IIA) of the periodic table: beryllium (Be), magnesium (Mg),calcium (Ca), strontium (Sr), barium (Ba) and radium (Ra).

In an embodiment the alkalinising agent of step iii) according to themethod of the present invention is selected from the group consisting ofNaOH, KOH, Ca(OH)₂ and LiOH.

In a further embodiment the alkalinising agent of step iii) according tothe method has a concentration of 0.1 M to 2.0 M, or of 0.1 M to 1.5 M,or of 0.2 M to 1.75 M or of 0.2 M to 1.0 M, or of at least 0.1, 0.2, 0.3or 0.4 M, or of at most 2.0, 1.7, 1.5, 1.3, 1.0 or 0.75 M, respectively.

In a further embodiment the alkalinising agent of step iii) according tothe method is selected from the group consisting of NaOH and KOH.

In a further embodiment the alkalinising agent used in the methodaccording to the present invention has in the mixture of sample+reagenti)+reducing agent ii)+alkalinising agent iii) a final concentration of0.1 M to 0.6 M, or of 0.2 M to 0.5 M, or of at least 0.1 or 0.2 M, or ofat most 0.6 or 0.5 M, respectively.

In a further embodiment of the method the mixing ratio of the threereagents of steps i) ii) and iii), respectively, to a sample to beinvestigated is preferably between 1:3 and 3:1.

In an embodiment the sample of step a), the reagent of step i)containing one hydrogen carbonate salt and a substance capable ofreleasing hydrogen carbonate ions (HCO₃ ⁻) upon hydrolysis+the reducingagent of step ii)+the alkalinising agent of step iii) according to themethod might be added in any pipetting sequence. Upon mixing, step b)according to the method of the present invention may be in a furtherembodiment characterized in that it has at least for 10, 12, 15, or 20seconds a pH value of 9.5 to 14, further preferred step b) has uponmixing at least for 10, 12, 15, or 20 seconds a pH value of 10.5 to 14.

In an embodiment of the method the sample to be investigated of step a)is mixed in step b) with the reagent of step i) containing one hydrogencarbonate salt and a substance capable of releasing hydrogen carbonateions (HCO₃ ⁻) upon hydrolysis, the reducing agent of step ii), thealkalinising agent of step iii) and incubated. The incubation step b)can be as long as required. The incubation time is e.g. from 15 secondsto 24 h. In one embodiment the mixture of step b) according to themethod of the present invention is incubated for 1 to 60 minutes therebyreleasing vitamin D compound from vitamin D-binding protein.

The concentrations of the components of step b) according to the methodare easily selected by a person skilled in the art such that thespecified pH range and the desired concentrations of one hydrogencarbonate salt and a substance capable of releasing hydrogen carbonateions (HCO₃ ⁻) upon hydrolysis, the reducing agent and the alkalinisingagent, respectively, during the incubation with the sample to beinvestigated are appropriate to release vitamin D compound from vitaminD-binding protein.

Alkaline conditions result in the denaturation of vitamin D-bindingprotein and release of vitamin D present in the sample to beinvestigated. The concentration of the alkalinising agent has to besufficient to increase the pH of the “reagent mixture” (=a sample to beinvestigated+reagent composition according to the presentinvention+alkalinising agent) to at least pH 10.0, preferably to atleast pH 10.5, more preferably to at least 11.0 in the pre-treatmentreaction. The skilled artisan is aware, that the pH of the reagentmixture has to be measured at the time of mixture of the sample to beinvestigated+reagent composition according to the presentinvention+alkalinising agent. Due to the hydrolysis of the hydrogencarbonate salt and/or the substance capable of releasing hydrogencarbonate ions (HCO₃ ⁻) upon hydrolysis, the pH will be reduced in thereagent mixture (see FIG. 1 and Example 1.5).

The term “sample” as used herein refers to a biological sample obtainedfrom an individual for the purpose of evaluation in vitro. In themethods of the present invention, the sample to be investigated is in anembodiment a liquid sample. The sample may comprise in a furtherembodiment of the present invention any body fluid. In a furtherembodiment the sample to be investigated is blood, serum or plasma, withserum or plasma being most preferred. In a further embodiment the liquidsample is dried on a filter paper or membrane. In an embodiment thesample used herein refers to an aliquot of a sample obtained from anindividual.

The present invention in a further embodiment comprises an in vitromethod for measuring a vitamin D compound comprising the steps of (a)releasing a vitamin D compound from vitamin D-binding protein and (b)measuring the vitamin D compound released in step (a).

In a further embodiment the present invention comprises an in vitromethod for measuring vitamin D compound comprising the steps of (a)releasing a vitamin D compound bound to vitamin D-binding protein in asample of interest and (b) measuring the vitamin D compounds released instep (a).

In a further embodiment the present invention concerns an in vitromethod for measuring a vitamin D compound comprising the steps of a)providing a sample to be investigated, b) mixing the sample from step(a) with i) a reagent containing one hydrogen carbonate salt and asubstance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) uponhydrolysis, wherein the total concentration of hydrogen carbonate ions(HCO₃ ⁻) from the hydrogen carbonate salt and released from thesubstance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) is 0.1to 2.0 M, ii) a reducing agent, and iii) an alkalinising agent, therebyreleasing a vitamin D compound from vitamin D-binding protein, and c)measuring the vitamin D compound released in step (b).

In a further embodiment the present invention comprises an in vitromethod for measuring a vitamin D compound, wherein the vitamin Dcompound measured is selected from the group comprising25-hydroxyvitamin D₂, 25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₂,24,25-dihydroxyvitamin D₃ and C3-epi 25-hydroxyvitamin D.

In a further embodiment the present invention comprises an in vitromethod for measuring a vitamin D compound, wherein the vitamin Dcompound measured is selected from the group comprising25-hydroxyvitamin D₂, 25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₂and 24,25-dihydroxyvitamin D₃.

In a further embodiment the present invention comprises an in vitromethod for measuring a vitamin D compound, wherein the vitamin Dcompounds 25-hydroxyvitamin D₂ and/or 25-hydroxyvitamin D₃ aredetermined.

Reagent Composition:

In one embodiment the present invention concerns a reagent compositionfor the release of a vitamin D compound from vitamin D-binding proteinin a sample to be investigated, which contains one hydrogen carbonatesalt and a substance capable of releasing hydrogen carbonate ions (HCO₃⁻) upon hydrolysis in a concentration, wherein the total concentrationof hydrogen carbonate ions (HCO₃ ⁻) from the hydrogen carbonate salt andreleased from the substance capable of releasing hydrogen carbonate ions(HCO₃ ⁻) is 0.1 to 2.0 M, and a reducing agent.

In a further embodiment the present invention concerns a reagentcomposition for the release of a vitamin D compound from vitaminD-binding protein in a sample to be investigated, which contains onehydrogen carbonate salt and a substance capable of releasing hydrogencarbonate ions (HCO₃ ⁻) upon hydrolysis, wherein the total concentrationof hydrogen carbonate ions (HCO₃ ⁻) from the hydrogen carbonate salt andreleased from the substance capable of releasing hydrogen carbonate ions(HCO₃ ⁻) is 0.1 to 1.5 M, and a reducing agent.

In a further embodiment the present invention concerns a reagentcomposition for the release of a vitamin D compound from vitaminD-binding protein in a sample to be investigated, which contains onehydrogen carbonate salt and a substance capable of releasing hydrogencarbonate ions (HCO₃ ⁻) upon hydrolysis, wherein the total concentrationof hydrogen carbonate ions (HCO₃ ⁻) from the hydrogen carbonate salt andreleased from the substance capable of releasing hydrogen carbonate ions(HCO₃ ⁻) is 0.2 to 1.0 M, and a reducing agent.

In a further embodiment the present invention concerns a reagentcomposition for the release of a vitamin D compound from vitaminD-binding protein in a sample to be investigated, which contains onehydrogen carbonate salt and a substance capable of releasing hydrogencarbonate ions (HCO₃ ⁻) upon hydrolysis, wherein the total concentrationof hydrogen carbonate ions (HCO₃ ⁻) from the hydrogen carbonate salt andreleased from the substance capable of releasing hydrogen carbonate ions(HCO₃ ⁻) is at least 0.1, 0.2, 0.3 or 0.4 M, and a reducing agent.

In a further embodiment the present invention concerns a reagentcomposition for the release of a vitamin D compound from vitaminD-binding protein in a sample to be investigated, which contains onehydrogen carbonate salt and a substance capable of releasing hydrogencarbonate ions (HCO₃ ⁻) upon hydrolysis, wherein the total concentrationof hydrogen carbonate ions (HCO₃ ⁻) from the hydrogen carbonate salt andreleased from the substance capable of releasing hydrogen carbonate ions(HCO₃ ⁻) is at most 2.0, 1.7, 1.5, 1.3, 1.0 or 0.75 M, and a reducingagent.

In a further preferred embodiment the hydrogen carbonate salt in thereagent composition is selected from the group consisting of sodiumhydrogen carbonate, potassium hydrogen carbonate, ammonium hydrogencarbonate, calcium hydrogen carbonate and magnesium hydrogen carbonate.Further preferred the hydrogen carbonate salt in the reagent compositionis selected from the group consisting of sodium hydrogen carbonate,potassium hydrogen carbonate and ammonium hydrogen carbonate. Furtherpreferred the hydrogen carbonate salt in the reagent composition issodium hydrogen carbonate and/or potassium hydrogen carbonate.

In a further preferred embodiment the substance capable of releasinghydrogen carbonate ions (HCO₃ ⁻) upon hydrolysis in the reagentcomposition is a cyclic or non-cyclic carbonate ester or a hydroxylatedor halogenized derivative thereof, respectively.

In a further preferred embodiment the substance capable of releasinghydrogen carbonate ions (HCO₃ ⁻) upon hydrolysis in the reagentcomposition is a cyclic or non-cyclic carbonate ester or halogenizedderivative thereof, respectively. In a further preferred embodiment thecyclic or non-cyclic carbonate ester or the halogenized derivativethereof in the reagent composition is selected from the group consistingof ethylene carbonate, dimethyl carbonate, propylene carbonate, vinylenecarbonate, trimethylene carbonate, erythritol bis-carbonate, glycerol1,2-carbonate, 4-chloro-1,3-dioxolan-2-one,4,5-dichloro-1,3-dioxolan-2-one, 2,5-dioxahexanedioic acid dimethylester, 1,2 butylene carbonate, cis 2,3 butylene carbonate and trans 2,3butylene carbonate. Further preferred the cyclic or non-cyclic carbonateester or the halogenized derivative thereof in the reagent compositionis selected from the group consisting of ethylene carbonate, dimethylcarbonate, propylene carbonate, vinylene carbonate, trimethylenecarbonate, erythritol bis-carbonate, glycerol 1,2-carbonate,4-chloro-1,3-dioxolan-2-one and 4,5-dichloro-1,3-dioxolan-2-one. Furtherpreferred the cyclic or non-cyclic carbonate ester in the reagentcomposition is selected from the group consisting of ethylene carbonate,dimethyl carbonate, glycerol 1,2-carbonate, propylene carbonate,vinylene carbonate. Further preferred the cyclic or non-cyclic carbonateester in the reagent composition is selected from the group consistingof ethylene carbonate, dimethyl carbonate, glycerol 1,2-carbonate andpropylene carbonate. Further preferred the cyclic or non-cycliccarbonate ester in the reagent composition is selected from the groupconsisting of ethylene carbonate, dimethyl carbonate and glycerol1,2-carbonate.

The present invention concerns in a further embodiment a reagentcomposition characterized in that the reducing agent is selected fromthe group consisting of 2-Mercaptoethanol, 2-Mercaptoethylamine-HCl,TCEP, Cystein-HCl, Dithiothreitol (DTT), N-Methylmaleimide, Ellman'sReagent and 1,2-dithiolane-3-carboxylic acid.

In a further embodiment the present invention concerns a reagentcomposition characterized in that the reducing agent contains thiolgroups.

In a further embodiment the present invention concerns a reagentcomposition characterized in that the reducing agent is selected fromthe group consisting of 2-Mercaptoethanol, 2-Mercaptoethylamine-HCl,TCEP, Cystein-HCl and Dithiothreitol (DTT).

The concentration of a reducing agent in a certain embodiment of thepresent invention is from 2 mM to 30 mM, in a further embodiment from 3mM to 20 mM, in a further embodiment from 3.5 mM to 15 mM and in afurther embodiment from 4 mM to 10 mM.

It is known to the person skilled in the art, that the capability of areducing agent is dependent on the presence of functional, i.e.,reducing groups. Therefore it is known to skilled artisan to select theappropriate concentration of a reducing agent taking into account it'snumber of active reducing groups.

The gene coding for the vitamin D-binding protein occurs in the humanpopulation in the form of different alleles. It is known that thepolypeptides coded by these alleles differ biochemically i.e. they leadto different phenotypes. These biochemical differences also influencethe binding and release of vitamin D compounds. The reagent compositionaccording to the invention is suitable for releasing vitamin D compoundsindependently of the phenotype of the vitamin D-binding protein. Thus apreferred embodiment of the present invention is the use of a reagentcomposition according to the invention to release vitamin D compoundsfrom vitamin D-binding protein.

The reagent composition according to the invention in one embodiment isused to release vitamin D compounds from vitamin D-binding protein insamples to be investigated irrespective and independent of thephenotypes of vitamin D-binding protein.

For the purpose of releasing vitamin D compounds from vitamin D-bindingprotein, the reagent composition according to the invention is mixedwith a sample to be investigated, e.g. serum or plasma, and analkalinising agent.

Reagent Mixture:

The term “reagent mixture” as used herein below comprises a sample to beinvestigated, a reagent composition according to the present invention,and an alkalinising agent.

In a further embodiment the reagent mixture is characterized in that thealkalinising agent is selected from the group consisting of NaOH, KOH,Ca(OH)₂ and LiOH.

In a further embodiment the reagent mixture is characterized in that theused alkalinising agent has a concentration of 0.1 M to 2.0 M, or of 0.1M to 1.5 M, or of 0.2 M to 1.75 M, or of 0.2 M to 1.0 M, or of at least0.1, 0.2, 0.3 or 0.4 M, or of at most 2.0, 1.7, 1.5, 1.3, 1.0 or 0.75 M,respectively.

In a further embodiment the reagent mixture is characterized in that thealkalinising agent is selected from the group consisting of NaOH andKOH.

In a further embodiment the alkalinising agent used in the methodaccording to the present invention has in the reagent mixture a finalconcentration of 0.1 M to 0.6 M, or of 0.2 M to 0.5 M, or of at least0.1, or 0.2 M, or of at most 0.6, or 0.5 M, respectively.

The mixing ratio of reagent composition and alkalinising agent to asample to be investigated is in an embodiment preferably between 1:3 and3:1.

A sample to be investigated, the reagent composition disclosed and analkalinising agent might be added in any pipetting sequence to form thereagent mixture. Upon mixing, the reagent mixture is in a furtherembodiment characterized in that it has at least for 10, 12, 15, or 20seconds a pH value of 9.5 to 14, further preferred the reagent mixturehas upon mixing at least for 10, 12, 15, or 20 seconds a pH value of10.5 to 14.

The sample to be investigated is mixed with the reagent compositionaccording to the invention and an alkalinising agent and incubated. Thisstep may also be called pre-treatment step. The pre-treatment step canbe performed as long as required. The incubation time is e.g. for 15seconds to 24 h. The reagent mixture in one embodiment is incubated for1 to 60 minutes to release vitamin D compounds from vitamin D-bindingprotein. The reagent mixture in another embodiment is incubated for 4 to10 minutes to release vitamin D compounds from vitamin D-bindingprotein.

The reagent mixture and concentrations of the components in it areeasily selected by a person skilled in the art such that the specifiedpH range and the desired concentrations of one hydrogen carbonate saltand a substance capable of releasing hydrogen carbonate ions (HCO₃ ⁻)upon hydrolysis, the reducing agent and the alkalinising agent,respectively, during the incubation with a sample to be investigated areappropriate to release vitamin D compounds from vitamin D-bindingprotein.

The reagent mixture comprises in an embodiment also the preferredsubstances and/or concentrations of one hydrogen carbonate salt and asubstance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) uponhydrolysis as described for the reagent composition of the presentinvention.

The detection of a vitamin D compound is preferably carried out suchthat at least one vitamin D compound selected from the group comprising25-hydroxyvitamin D₂, 25-hydroxyvitamin D₃, 24,25 dihydroxyvitamin D₂,24,25-dihydroxyvitamin D₃ and C3-epi 25-hydroxyvitamin D is detected.

In the specific detection of a vitamin D compound further incubationsteps follow after the pre-treatment step. The leftover of the reducingagent present in the reagent mixture can be blocked by addition ofunspecific proteins, preferably e.g. human serum albumin (HSA). Thisunspecific proteins can be added separately or can be simply included inthe solution also comprising the detecting reagent. By blocking theresidual reducing capability of the reducing agent, a noncompromiseddetection of a vitamin D compound using a proteinaceous specific bindingagent to a vitamin D compound is possible.

As the person skilled in the art will appreciate, the solutioncomprising the specific binding agent will contain a pH buffer systemwhich ensures after addition of the solution containing the specificbinding agent to the reagent mixture the pH is a prerequisite forbinding of a vitamin D compound to the specific binding agent. Neitherthe necessarily required buffer system nor the final pH are critical aslong as binding of the specific binding agent to a vitamin D compoundtakes place. In case that vitamin D-binding protein is used as aspecific binding agent, the pH during this incubation step is preferablyselected between pH 6.0 and pH 9.0. In case that an antibody is used asa specific binding agent for a vitamin D compound, the pH during thisincubation step preferably will be between pH 5.5 and pH 7.5.

The solution comprising the specific binding agent preferably contains abuffer system that is 20 mM to 400 mM. Also preferred the buffer has amolarity of between 50 mM and 350 mM or between 100 mM and 300 mM.

The in vitro method for the detection of a vitamin D compound can—basedon the disclosure of the present invention—be carried out in variousways.

In principle all proteinaceous binding partners such as specificallybinding polypeptides that bind to one or more vitamin D compound can beused as a specific binding agent. A specific binding agent can be eitheran antibody or vitamin D-binding protein itself.

Many commercial test systems are based on the use of solid phases coatedwith avidin or streptavidin (SA), for example SA-coated microtitreplates or SA-coated latices.

A biotinylated analyte derivative is for example bound to this SA solidphase before or during the test procedure. When detecting vitamin Dcompound this biotinylated analyte derivative compound can for examplebe a biotinylated 25-hydroxyvitamin D₂ and/or a biotinylated25-hydroxyvitamin D₃.

In one embodiment of the present invention the in vitro method ofdetection is carried out as a competitive assay. In such a competitivetest a derivative of vitamin D compound added in a defined amount to thetest competes with the corresponding vitamin D compound from the samplefor the binding sites of the specific binding agent. The more vitamin Dcompound is present in the sample, the smaller is the detection signal.

In one embodiment the derivative of a vitamin D compound is abiotinylated vitamin D compound. In a further embodiment thebiotinylated vitamin D compound is a biotinylated 25-hydroxyvitamin D₂and/or biotinylated 25-hydroxyvitamin D₃. In a further embodiment thebiotinylated vitamin D compound is a biotinylated 25-hydroxyvitamin D₂.

As mentioned above preferred specific binding agents for use in adetection method as disclosed in the present description are antibodiesand vitamin D-binding protein. Vitamin D-binding protein, if used in acompetitive assay format, will lead to an integrated measurement of allvitamin D compounds competing with its binding to one ore more(biotinylated) vitamin D compound derivative. In one embodiment thevitamin D-binding protein will be detectable labelled, e.g.ruthenylated.

Use:

In one embodiment the present invention relates to the use of a reagentcomposition together with an alkalinising agent to release a vitamin Dcompound from vitamin D-binding protein.

In a further embodiment the present invention relates to the use of areagent composition together with an alkalinising agent to release avitamin D compound expected to be present in a sample to be investigatedfrom vitamin D-binding protein.

In a further embodiment the present invention relates to the use of areagent composition together with an alkalinising agent to release avitamin D compound in method of detecting a vitamin D compound.

In a further embodiment the present invention relates to the use of areagent composition containing one hydrogen carbonate salt and asubstance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) uponhydrolysis, wherein the total concentration of hydrogen carbonate ions(HCO₃ ⁻) from the hydrogen carbonate salt and released from thesubstance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) is 0.1 Mto 2.0 M, 2 mM to 30 mM of a reducing agent, together with a solution of1 M to 1.5 M of an alkalinising agent to release a vitamin D compoundexpected to be present in a sample to be investigated from vitaminD-binding protein in method of detecting a vitamin D compound.

The use of the reagent composition comprises in an embodiment also thepreferred substances and/or concentrations of one hydrogen carbonatesalt and a substance capable of releasing hydrogen carbonate ions (HCO₃⁻) upon hydrolysis as described for the reagent composition of thepresent invention.

Kit:

In one embodiment the present invention relates to a kit for the releaseof a vitamin D compound from vitamin D-binding protein, which contains areagent composition comprising one hydrogen carbonate salt and asubstance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) uponhydrolysis, wherein the total concentration of hydrogen carbonate ions(HCO₃ ⁻) from the hydrogen carbonate salt and released from thesubstance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) is 0.1 Mto 2.0 M, and a reducing agent.

In one embodiment the present invention relates to a kit for thedetection of a vitamin D compound from vitamin D-binding protein,characterized in that it comprises a reagent composition which has onehydrogen carbonate salt and a substance capable of releasing hydrogencarbonate ions (HCO₃ ⁻) upon hydrolysis, wherein the total concentrationof hydrogen carbonate ions (HCO₃ ⁻) from the hydrogen carbonate salt andreleased from the substance capable of releasing hydrogen carbonate ions(HCO₃ ⁻) is 0.1 M to 2.0 M, a reducing agent, and an alkalinising agent.

In a further embodiment the present invention relates to a kit for thedetection of a vitamin D compound from vitamin D-binding protein,characterized in that it comprises a reagent composition which has onehydrogen carbonate salt and a substance capable of releasing hydrogencarbonate ions (HCO₃ ⁻) upon hydrolysis, wherein the total concentrationof hydrogen carbonate ions (HCO₃ ⁻) from the hydrogen carbonate salt andreleased from the substance capable of releasing hydrogen carbonate ions(HCO₃ ⁻) is 0.1 M to 2.0 M, 2 mM to 30 mM of a reducing agent, and analkalinising agent.

In a further embodiment the present invention relates to a kit for thedetection of a vitamin D compound from vitamin D-binding protein,characterized in that it comprises a reagent composition which has onehydrogen carbonate salt and a substance capable of releasing hydrogencarbonate ions (HCO₃ ⁻) upon hydrolysis, wherein the total concentrationof hydrogen carbonate ions (HCO₃ ⁻) from the hydrogen carbonate salt andreleased from the substance capable of releasing hydrogen carbonate ions(HCO₃ ⁻) is 0.1 M to 2.0 M, 2 mM to 30 mM of a reducing agent, asolution of 1 M to 1.5 M of an alkalinising agent, in addition to thedetecting components.

In a further embodiment the present invention relates to a kit for thedetection of a vitamin D compound characterized in that it comprises areagent composition which has one hydrogen carbonate salt and asubstance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) uponhydrolysis, a reducing agent, a solution of an alkalinising agent, inaddition to a solution comprising a specific binding agent.

In a further embodiment the present invention relates to a kit for thedetection of a vitamin D compound characterized in that it comprises areagent composition which has one hydrogen carbonate salt and asubstance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) uponhydrolysis, wherein the total concentration of hydrogen carbonate ions(HCO₃ ⁻) from the hydrogen carbonate salt and released from thesubstance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) is 0.1 Mto 2.0 M, 2 mM to 30 mM of a reducing agent, a solution of 1 M to 1.5 Mof an alkalinising agent and a solution comprising a specific bindingagent.

In a further embodiment the present invention relates to a kit for thedetection of a vitamin D compound characterized in that it comprises areagent composition which has one hydrogen carbonate salt and asubstance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) uponhydrolysis, wherein the total concentration of hydrogen carbonate ions(HCO₃ ⁻) from the hydrogen carbonate salt and released from thesubstance capable of releasing hydrogen carbonate ions (HCO₃ ⁻) is 0.1 Mto 2.0 M, 2 mM to 30 mM of a reducing agent selected from the groupconsisting of 2-Mercaptoethanol, 2-Mercaptoethylamine-HCl, TCEP,Cystein-HCl and Dithiothreitol (DTT), a solution of 1 M to 1.5 M of analkalinising agent selected from the group consisting of NaOH, KOH,Ca(OH)₂ and LiOH and a solution comprising a specific binding agent.

The kit comprises in an embodiment also the preferred substances and/orconcentrations of one hydrogen carbonate salt and a substance capable ofreleasing hydrogen carbonate ions (HCO₃ ⁻) upon hydrolysis as describedfor the reagent composition of the present invention.

The reagent composition according to the invention has proven to besuitable for use in an automated test for vitamin D compounds. Thepresent invention preferably concerns the use of a reagent compositionaccording to the invention for releasing vitamin D compounds fromvitamin D-binding protein especially in a test for the determination ofvitamin D compounds.

The test for a vitamin D compound is preferably completely automated.Completely automated in this case means that the experimentator only hasto place a sample to be investigated and a reagent pack containing allcomponents for measuring a vitamin D compound on an automated analyzerand all further steps are carried out automatically by the analyzer. Thecompletely automated test is particularly preferably carried out on anElecsys® analyzer from Roche Diagnostics.

The reagent composition according to the invention in a furtherembodiment is used in an in vitro method for the detection of a vitaminD compound selected from the group comprising 25-hydroxyvitamin D₂,25-hydroxyvitamin D₃, 24,25 dihydroxyvitamin D₂, 24,25-dihydroxyvitaminD₃ and C3-epi 25-hydroxyvitamin D.

As already mentioned above 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃are particularly relevant forms of vitamin D for diagnostics. In the invitro method according to the invention the specific detection of25-hydroxyvitamin D₂ or 25-hydroxyvitamin D₃ or both via a specificantibody to 25-hydroxyvitamin D₂ or 25-hydroxyvitamin D₃ also representsa preferred embodiment.

The invention is further elucidated by the following examples andfigures. The actual protective scope results from the claims attached tothis invention.

DESCRIPTION OF THE FIGURES

FIG. 1: pH change of the reagent mixture during the pre-treatment step.The assay was performed as outlined in example 1.5. Reagent composition(A) contains various concentrations of ethylene carbonate (EC): 0.00 M(), 0.10 M (♦), 0.30 M (□), 0.50 M (▴), 0.75 M (◯), 1.00 M (▪), 1.50 M(⋄) EC. The X axis shows the time in minutes, the Y axis the pH.

FIG. 2: Calibration curves of a Vitamin D assay as described in example1.5 with reagent composition (A) containing various concentrations ofethylene carbonate (EC): 1.50 M (), 1.00 M (□), 0.75 M (♦), 0.50 M (◯),0.30 M (▴) and 0.10 M (⋄) EC. The X axis shows the concentration inng/ml, the Y axis shows the counts determined as usual using theElecsys® system from the Roche Diagnostics company.

FIG. 3: Calibration curves of a Vitamin D assay as described in example1.5 with reagent composition (A) containing various concentrations ofthe reducing agent dithiothreitol (DTT): 1.0 mM (□), 2.0 mM (), 4.0 mM(♦), 6.7 mM (◯), 10.0 mM/12.0 mM (▴), 15.0 mM (⋄). The X axis shows theconcentration in ng/ml, the Y axis shows the counts determined as usualusing the Elecsys® system from the Roche Diagnostics company.

FIG. 4a : Method comparison: Vitamin D assay (example 1) and liquidchromatography-tandem mass spectrometry (LC-TMS) 25-hydroxyvitamin D wasdetermined by means of LC-TMS as well as by means of the vitamin D assayof example 1.5, where reagent composition (A) with 0.5 M ethylenecarbonate (EC) was used for the incubation. The results in ng/ml formultiple serum samples are plotted on the X axis for the LC-TMS and onthe Y axis for the vitamin D assay of example 1.5.

-   -   (---) y=x    -   (—) Linear regression        -   Vitamin D assay=2.0116+0.9036*x, Pearsons r=0.9509

FIG. 4b : Method comparison: Vitamin D assay (example 1) and LC-TMS25-hydroxyvitamin D was determined by means of LC-TMS as well as bymeans of the vitamin D assay of example 1.5, where reagent composition(A) without ethylene carbonate (EC) was used for the incubation. Theresults in ng/ml for multiple serum samples are plotted on the X axisfor the LC-TMS and on the Y axis for the vitamin D assay of example 1.5.

-   -   (---) y=x    -   (—) Linear regression        -   Vitamin D assay=0.7496+0.7338*x, Pearsons r=0.7914

FIG. 5: Calibration curves of a Vitamin D assay as described in example2 with reagent composition (A) containing 0.5 M ethylene carbonate (♦),0.5 M Na₂CO₃ (◯), 0.5 M NaH₂PO₄ (▴), 0.5 M NaCl (⋄), and control (□).The X axis shows the concentration in ng/ml, the Y axis shows the countsdetermined as usual using the Elecsys® system from the Roche Diagnosticscompany.

FIG. 6: Calibration curves of a Vitamin D assay as described in example3 with reagent composition (A) containing:

-   -   ♦: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M EC, or    -   ▴: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, or    -   □: 10 mM NaOH, 4 mM EDTA.    -   The X axis shows the concentration in ng/ml, the Y axis shows        the counts determined as usual using the Elecsys® system from        the Roche Diagnostics company.

FIG. 7: Calibration curves of a Vitamin D assay as described in example4 with reagent composition (A) containing:

-   -   ◯: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M EC (see example        1.5), or    -   ♦: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M dimethyl carbonate.    -   The X axis shows the concentration in ng/ml, the Y axis shows        the counts determined as usual using the Elecsys® system from        the Roche Diagnostics company.

FIG. 8: Calibration curves of a Vitamin D assay as described in example5 with reagent composition (A) containing:

-   -   ♦: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M EC (see example        1.5), or    -   ◯: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M NaHCO₃, or    -   ▴: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M NaHCO₃+0.5 M        ethylene glycol, or    -   □: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT.    -   The X axis shows the concentration in ng/ml, the Y axis shows        the counts determined as usual using the Elecsys® system from        the Roche Diagnostics company.

FIG. 9: Calibration curves of a Vitamin D assay as described in example4 with reagent composition (A) containing:

-   -   ♦: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M EC (see example 1.5)        or    -   ◯: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M glycerol 1,2        carbonate.    -   The X axis shows the concentration in ng/ml, the Y axis shows        the counts determined as usual using the Elecsys® system from        the Roche Diagnostics company.

EXAMPLE 1 Assays for the Detection of 25-Hydroxyvitamin D

Commercial assays are used according to the manufacturer's instructions.The 25-hydroxyvitamin D determinations are carried out by means of HPLC(test for 25(OH)vitamin D₃, from the “Immundiagnostik” Company,Bensheim, order No. KC 3400) or by means of LC-MS/MS (Vogeser, M. etal., Clin. Chem. 50 (2004) 1415-1417) as described in the literature.

The preparation of the ingredients and the general test procedure for anew test is described in the following:

1.1 Synthesis of hydroxyvitamin D2-3-2′-cyanoethyl ether

20.6 mg (50 μmol) 25-hydroxyvitamin D₂ (Fluka No. 17937) is dissolved ina 25 ml three necked round bottom flask with an internal thermometer in10 ml dry acetonitrile under an argon atmosphere. 1.5 mltert.-butanol/acetonitrile (9:1) is added to the solution and cooled to6° C. in an ice bath. Subsequently 820 μl of an acrylonitrile solution(86 μl acrylonitrile in 1.0 ml acetonitrile) is added and stirred for 15minutes at 6° C. Then 205 μl of a potassium hydride solution (25 mg KHin 0.5 ml tert.-butanol/acetonitrile 9:1) is added. A brief flocculationoccurs after which a clear solution is obtained. The reaction solutionis stirred for a further 45 minutes at 6° C. and subsequently for 60minutes at 4° C.

Subsequently the reaction solution is diluted with 10 mlmethyl-tert.-butyl ether and washed twice with 10 ml H₂O each time. Theorganic phase is dried with about 1 g anhydrous sodium sulfate, filteredover a G3 glass frit and evaporated on a rotary evaporator. It is driedin a high vacuum to form a viscous clear residue with a mass of about 55mg.

1.2 Synthesis of hydroxyvitamin D₂-3-3-aminopropyl ether

The entire nitrile obtained above is dissolved in 15 ml diethyl etherand admixed with a suspension of 7.5 mg lithium hydride in 7.5 mldiethyl ether while stirring. The reaction mixture is stirred for 1 hourat room temperature. Afterwards a suspension of 38.4 lithium aluminiumhydride in 6.6 ml diethyl ether is added. This results in a strongturbidity of the mixture. The reaction mixture is stirred for a furtherhour at room temperature, then the reaction mixture is cooled to 0-5° C.in an ice bath and 35 ml water is carefully added. The pH is madestrongly basic by addition of 6.6 ml 10 M potassium hydroxide solution.

It is extracted three times with 65 ml methyl-tert.-butyl ether eachtime. The combined organic phases are dried using about 5 g anhydroussodium sulfate, filtered and evaporated at room temperature on a rotaryevaporator. The residue is dried to mass constancy using an oil pump.The crude product is dissolved in 5 ml DMSO and 3.0 ml acetonitrile andpurified by means of preparative HPLC.

eluent A=Millipore-H₂O+0.1% trifluoroacetic acid;eluent B=95% acetonitrile+5% Millipore-H₂O+0.1% TFA;gradient: from 50% B to 100% B in 100 minflow rate: 30 ml/mintemperature: room temperaturecolumn dimension: Ø=5.0 cm; L=25 cmcolumn material: Vydac C18/300 Å/15-20 μmdet. wavelength: 226 nm

Fractions whose product content is larger than 85% according toanalytical HPLC (Vydac C18/300 Å/5 μm; 4.6×250 mm) are pooled in a roundbottom flask and lyophilized. 13.7 mg (yield: 58%) is obtained as acolourless lyophilisate.

1.3 Synthesis of hydroxyvitaminD2-3-3′-N-(hemisuberyl)aminopropyl-ether-biotin-(beta-Ala)-Glu-Glu-Lys(epsilon)conjugate (=Ag—Bi)

13.7 mg (25 μmol) hydroxyvitamin D₂-3-3′-aminopropyl ether is dissolvedin 3.5 ml DMSO, 28.7 mg (30 μmol)biotin-(beta-Ala)-Glu-Glu-Lys(epison)-hemi-suberate-N-hydroxysuccinimideester (Roche Applied Science, No. 11866656) and 12.5 μl triethylamineare added and it is stirred overnight at room temperature. The reactionsolution is diluted with 4.5 ml DMSO, filtered through a 0.45 μmmicrofilter and subsequently purified by means of preparative HPLC(conditions see example 2.3 b)). Fractions that contain more than 85%product according to analytical HPLC are pooled and lyophilized. 9.8(yield: 30%) purified biotin conjugate is obtained.

1.4 Ruthenylation of Vitamin D-Binding Protein and Purification by GelFiltration Chromatography

The vitamin D-binding protein is transferred to 100 mM potassiumphosphate/150 mM sodium chloride buffer, pH 8.5 and the proteinconcentration is adjusted to 5-10 mg/ml. The ruthenylation reagent(ruthenium (II) tris (bipyridyl)-N-hydroxysuccinimide ester) isdissolved in DMSO and added to the antibody solution at a molar ratio of3 to 1. After a reaction time of 45 min the reaction is stopped byaddition of 1-lysine and the ruthenylated vitamin D-binding protein(=DBP-Ru) is purified by gel filtration on a Superdex 200 column.

1.5 Test Procedure in the Assay

The sample to be investigated is measured using the Elecsys® system fromthe Roche Diagnostics company.

The reagent mixture is formed by mixing a sample to be investigated withthe reagent composition (A) and an alkalinising agent (B).

In this example the reagent mixture is formed of 15 μl sample mixed with15 μl of the reagent composition (A) and 10 μl of the alkalinising agent(B). The reagent mixture is incubated for 9 minutes. In the next step 70μl of detecting reagent (Solution C) is added to the reagent mixture andincubated for further 9 minutes. In the last step biotinylated wallantigen (Solution D) (60 μl) as well as 30 μl of magnetizablepolystyrene particles coated with streptavidin (SA) (30 μl) (SuspensionE) are added. After a further 9 minutes incubation the amount of boundruthenylated vitamin D-binding protein is determined as usual (see FIG.1, 2, 3, 4 a, 4 b).

Reagent composition (A) contains:

10 mM NaOH  4 mM EDTA 6.7 mM  dithiothreitol (DTT) 0.5M ethylenecarbonate (EC) pH 5.5

Alkalinising agent (B) contains:

1.375M NaOH

Solution C with the ruthenylated vitamin D-binding protein (DBP-Ru)contains:

0.2M bis-tris-propane (pH 7.5) 2.5% human serum albumin (HSA) 50 mM NaCl 1% mannit 0.1% oxypyrion 0.12 μg/mL DBP-Ru

Solution D with the biotinylated wall antigen contains:

0.2M bis-tris-propane (pH 8.6) 0.5% tween-20 solution 0.1% oxypyrion 30ng/ml biotin 0.0108 μg/mL Ag—Bi (from example 1.1)

Suspension E with SA-coated latex particles contains:

0.72 mg/ml SA-coated magnetizable polystyrene particles having a bindingcapacity of 470 ng/ml.

EXAMPLE 2 Comparison of Carbonate Ester to a Metal Salt, a PhosphateBuffer and a Carbonate

The sample to be investigated is measured using the Elecsys® system fromthe Roche Diagnostics company. The total assay procedure is shown inexample 1.5.

In aberrance to example 1.5 the reagent composition (A) contains either0.5 M ethylene carbonate (EC), 0.5 M Na₂CO₃, 0.5 M NaCl or 0.5 MNaH₂PO₄, respectively.

Reagent composition (A):

10 mM NaOH  4 mM EDTA 6.7 mM  DTT 0.5M of either EC, Na₂CO₃, NaCl orNaH₂PO₄

As control a reagent composition (A) containing 10 mM NaOH, 4 mM EDTA,6.7 mM DTT has been used. The results are shown in FIG. 5. The carbonateester (0.5 M EC (♦) present in the alkaline pretreatment (reagentmixture) causes a signal enhancing effect in the competitive assay.Especially the signal dynamic is improved compared to a test without EC(□). A salt (0.5 M NaCl, (⋄)) shows no effect. The addition of 0.5 MNa₂CO₃ (◯) or 0.5 M NaH₂PO₄ (▴) shows a minor effect on the signal.

EXAMPLE 3 Alkaline Pretreatment with/without Carbonate Ester

The sample to be investigated is measured using the Elecsys® system fromthe Roche Diagnostics company. The assay procedure is shown in example1.5.

In aberrance to example 1.5 three different reagent compositions havebeen prepared containing either:

♦: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M EC (see example 1.5) or

▴: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT or □: 10 mM NaOH, 4 mM EDTA.

After a 4 min pretreatment incubation of sample+either ♦ (reagentcomposition (A)+alkalinising agent (B) as described in example 1.5), ▴,or □, respectively, (=reagent mixture) and before addition of solution Cthe pH of the reagent mixture has been set to pH 9 by addition ofbis-tris-propane pH 6.3 (FIG. 6). The carbonate ester EC present in thealkaline pretreatment (reagent mixture) causes a signal enhancing effectin the competitive assay. Especially the signal dynamic is improvedcompared to a test without EC.

EXAMPLE 4 Ethylene Carbonate Vs Dimethyl Carbonate

The sample to be investigated is measured using the Elecsys® system fromthe Roche Diagnostics company. The assay procedure is shown in example1.5.

In aberrance to example 1.5 two different reagent compositions (A) havebeen prepared containing either:

◯: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M EC (see example 1.5) or♦: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M dimethyl carbonate.

Both carbonate ester, ethylene carbonate or dimethyl carbonate,respectively, show the same assay performance (FIG. 7).

EXAMPLE 5 Effect of the Hydrolysis Products of Ethylene Carbonate

The sample to be investigated is measured using the Elecsys® system fromthe Roche Diagnostics company. The assay procedure is shown in example1.5.

In aberrance to example 1.5 five different reagent compositions (A) havebeen prepared containing either:

♦: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M EC (see example 1.5) or

◯: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M NaHCO₃ or

▴: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M NaHCO₃+0.5 M ethylene glycol

□: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT.

The alkaline hydrolysis product of EC is ethylene glycol, which has noinfluence on the assay (♦). A hydrogene carbonate salt (NaHCO₃) showsalso a signal enhancing effect, but not as much as a carbonate ester(FIG. 8).

EXAMPLE 6 Ethylene Carbonate Vs Glycerol 1,2 Carbonate

The sample to be investigated is measured using the Elecsys® system fromthe Roche Diagnostics company. The assay procedure is shown in example1.5.

In aberrance to example 1.5 two different reagent compositions (A) havebeen prepared containing either:

♦: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M EC (see example 1.5) or◯: 10 mM NaOH, 4 mM EDTA, 6.7 mM DTT, 0.5 M glycerol 1,2 carbonate.

Both carbonate ester, ethylene carbonate or glycerol 1,2 carbonate,respectively, show the same assay performance (FIG. 9).

1. An in vitro method for releasing a vitamin D compound from vitaminD-binding protein comprising the step of: a) providing a sample to beinvestigated and b) mixing the sample from step (a) with i) a reagentcontaining one hydrogen carbonate salt and a substance capable ofreleasing hydrogen carbonate ions (HCO₃ ⁻) upon hydrolysis, wherein thetotal concentration of hydrogen carbonate ions (HCO₃ ⁻) from thehydrogen carbonate salt and released from the substance capable ofreleasing hydrogen carbonate ions (HCO₃ ⁻) is 0.1 M to 2.0 M, ii) areducing agent, and iii) an alkalinising agent, thereby releasing thevitamin D compound from vitamin D-binding protein.
 2. The methodaccording to claim 1, wherein the reagent according to step (i) issoluble in an aqueous solution under the appropriate conditions forreleasing a vitamin D compound from vitamin D-binding protein.
 3. Themethod according to claim 1, wherein the substance capable of releasinghydrogen carbonate ions (HCO₃ ⁻) upon hydrolysis is a cyclic ornon-cyclic carbonate ester or a hydroxylated or halogenized derivativethereof, respectively.
 4. The method according claim 1, wherein thesample is a liquid sample.
 5. The method according to claim 1, whereinthe sample is blood, serum or plasma.
 6. An in vitro method formeasuring a vitamin D compound comprising the steps of: a) releasing avitamin D compound from vitamin D-binding protein according to themethod of claim 1; and b) measuring the vitamin D compound released instep (a).
 7. The method according to claim 6, wherein the vitamin Dcompound is selected from the group consisting of 25-hydroxyvitamin D₂,25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₂, 24,25-dihydroxyvitaminD₃ and C3-epi 25-hydroxyvitamin D.
 8. The method according to claim 7,wherein the vitamin D compounds 25-hydroxyvitamin D₂ and/or25-hydroxyvitamin D₃ are determined.
 9. A reagent composition for therelease of a vitamin D compound from vitamin D-binding proteincomprising one hydrogen carbonate salt and a substance capable ofreleasing hydrogen carbonate ions (HCO₃ ⁻) upon hydrolysis, wherein thetotal concentration of hydrogen carbonate ions (HCO₃ ⁻) from thehydrogen carbonate salt and released from the substance capable ofreleasing hydrogen carbonate ions (HCO₃ ⁻) is 0.1 M to 2.0 M, and areducing agent.
 10. The reagent composition according to claim 9,characterized in that the reducing agent is selected from the groupconsisting of 2-Mercaptoethanol, 2-Mercaptoethylamine-HCl, TCEP,Cystein-HCl, Dithiothreitol (DTT), N-Methylmaleimide, Ellman's Reagentand 1,2-dithiolane-3-carboxylic acid.
 11. The reagent compositionaccording to claim 9, characterized in that the reducing agent isselected from the group consisting of 2-Mercaptoethanol,2-Mercaptoethylamine-HCl, TCEP, Cystein-HCl and Dithiothreitol (DTT).12. The reagent composition according to claim 9, characterized in thatthe reducing agent has a concentration of 2 mM to 30 mM.
 13. A reagentmixture comprising a sample to be investigated, a reagent compositionaccording to claim 9, and an alkalinising agent selected from the groupconsisting of NaOH, KOH, Ca(OH)₂ and LiOH, wherein the sample is blood,serum or plasma.